Maternal biomarkers for gestational diabetes

ABSTRACT

Embodiments herein relate to the field of screening tools for fetal/maternal wellness, and, more specifically, to biomarkers for gestational diabetes. In various embodiments, the methods may provide non-invasive and minimally-invasive screening tools for gestational diabetes that involve detection of changes in a proteomic profile of a test sample relative to a reference sample. In particular embodiments, the method may include determining whether a proteomic profile of a test sample from the subject includes at least one expression signature characteristic of gestational diabetes, wherein the proteomic profile comprises information on the expression of glycosylated fibronectin and glycosylated PSG, for example information on levels of fibronectin-SNA or a fibronectin-antibody complex, and PSG-AAL or a PSG-antibody complex. In some embodiments, the proteomic profile may also include information on the expression of adiponectin, sex hormone binding globulin (SHBG), C-reactive protein (CRP), a ratio of human chorionic gonadotropin (hCG) to placental lactogen, or a combination thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/085,974, filed Nov. 21, 2013, entitled “MATERNAL BIOMARKERSFOR GESTATIONAL DIABETES,” which, in turn, was a divisional of U.S.patent application Ser. No. 13/792,844, filed Mar. 11, 2013, entitled“MATERNAL BIOMARKERS FOR GESTATIONAL DIABETES,” which claims priority toU.S. Provisional Patent Application No. 61/623,690, filed Apr. 13, 2012,entitled “MATERNAL BIOMARKERS FOR GESTATIONAL DIABETES,” the entiredisclosure of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

Embodiments herein relate to the field of screening tools forfetal/maternal wellness, and, more specifically, to biomarkers forgestational diabetes.

BACKGROUND

The rates of obesity and diabetes have increased rapidly over the last20 years, both in the United States and globally. The incidence ofgestational diabetes mellitus (GDM) is also increasing, paralleling theoverall rise in obesity and type-2 diabetes. The adoption of newdiagnostic criteria based upon the recent Hyperglycemia and AdversePregnancy Outcomes (HAPO) study is expected to increase the prevalenceof GDM to about 18% of all pregnancies. In light of the fact that 80-90%of women with GDM can be managed with lifestyle therapy alone, universalscreening for GDM is increasingly considered justified.

GDM is a serious complication of pregnancy that can increase the risk ofa number of maternal-fetal disorders, including macrosomia, shoulderdystocia or birth injury, premature delivery, and preeclampsia. Inaddition to the increased risk of complications associated withgestation and delivery, there are also serious post-natal complicationsassociated with GDM. For instance, 5 to 10% of women with GDM are foundto have diabetes immediately after pregnancy, and women who have had GDMhave a 10-fold higher chance of developing diabetes within the next10-20 years. Children of mothers with GDM have an eight-fold greaterrisk of developing type-2 diabetes in later life. Thus, untreated GDMcontributes to the overall diabetic population in both the short andlong term.

Universal or even widespread GDM screening is hampered by the fact thatthe standard assessments of diabetes and pre-diabetes, such as fastinginsulin/glucose and HbA1c, are not recommended for screening of GDM.Instead, the recommended parameter is an oral glucose tolerance test(OGTT), which is costly and invasive, requiring a hospital visit andmultiple blood draws.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will be readily understood by the following detaileddescription in conjunction with the accompanying drawings. Embodimentsare illustrated by way of example and not by way of limitation in thefigures of the accompanying drawings.

FIG. 1 illustrates a two-dimensional differential in-gel electrophoresis(2D-DIGE) analysis of control and GDM maternal serum samples, in whichdifferentially abundant proteins (arrows) appear as red or green spotsdepending on the extent of under- or over-abundance, in accordance withvarious embodiments;

FIG. 2 illustrates receiver operating characteristic (ROC) curves thatdemonstrate the ability of fibronectin and pregnancy-specificglycoprotein (PSG) glycosylation to distinguish samples from pregnantwomen with and without gestational diabetes, in accordance with variousembodiments;

FIG. 3 illustrates ROC curves that demonstrate the classificationperformance of each protein and protein glycosylation pattern asindividual analytes and as a multi-analyte model, in accordance withvarious embodiments

FIG. 4 illustrates serial measures of fibronectin-SNA across trimestersin 35 non-diabetic controls, in which the lines represent the 25th and75th percentile of the GDM study group, in accordance with variousembodiments;

FIG. 5 is a Receiver Operating Characteristic (ROC) curve illustratingthe ability of first trimester fibronectin-SNA to predict subsequentdevelopment of gestational diabetes, in accordance with variousembodiments;

FIG. 6 is a Receiver Operating Characteristic (ROC) Curve, illustratingthe ability of fibronectin and PSG glycosylation to distinguish betweennon-diabetics and gestational diabetics within the second and thirdtrimesters of pregnancy, in accordance with various embodiments;

FIG. 7 is a Receiver Operating Characteristic (ROC) Curve, illustratingthe classification performance of each protein and protein glycosylationpattern as individual analytes and as a multi-analyte model, inaccordance with various embodiments;

FIGS. 8A and 8B illustrate a schematic diagram showing an example of alateral flow immunoassay (FIG. 8A) and a lateral flow device (FIG. 8B)that may be used in accordance with various embodiments disclosedherein; and

FIG. 9 is a graph illustrating a comparison of the results of afibronectin ELISA with the results of a FN-LFIA test, in accordance withvarious embodiments.

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

In the following detailed description, reference is made to theaccompanying drawings which form a part hereof, and in which are shownby way of illustration embodiments that may be practiced. It is to beunderstood that other embodiments may be utilized and structural orlogical changes may be made without departing from the scope. Therefore,the following detailed description is not to be taken in a limitingsense, and the scope of embodiments is defined by the appended claimsand their equivalents.

Various operations may be described as multiple discrete operations inturn, in a manner that may be helpful in understanding embodiments;however, the order of description should not be construed to imply thatthese operations are order dependent.

The description may use perspective-based descriptions such as up/down,back/front, and top/bottom. Such descriptions are merely used tofacilitate the discussion and are not intended to restrict theapplication of disclosed embodiments.

The terms “coupled” and “connected,” along with their derivatives, maybe used. It should be understood that these terms are not intended assynonyms for each other. Rather, in particular embodiments, “connected”may be used to indicate that two or more elements are in direct physicalor electrical contact with each other. “Coupled” may mean that two ormore elements are in direct physical or electrical contact. However,“coupled” may also mean that two or more elements are not in directcontact with each other, but yet still cooperate or interact with eachother.

For the purposes of the description, a phrase in the form “NB” or in theform “A and/or B” means (A), (B), or (A and B). For the purposes of thedescription, a phrase in the form “at least one of A, B, and C” means(A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C). For thepurposes of the description, a phrase in the form “(A)B” means (B) or(AB) that is, A is an optional element.

The description may use the terms “embodiment” or “embodiments,” whichmay each refer to one or more of the same or different embodiments.Furthermore, the terms “comprising,” “including,” “having,” and thelike, as used with respect to embodiments, are synonymous.

Disclosed herein in various embodiments are non-invasive and minimallyinvasive methods that may be used for the widespread screening forgestational diabetes. In various embodiments, the methods may includedetermining whether a proteomic profile of a test sample from a subjectincludes at least one expression signature characteristic of gestationaldiabetes, wherein the proteomic profile includes information on theexpression of glycosylated fibronectin, for example by detection and/orquantification of glycosylated fibronectin, such bydetecting/quantifying fibronectin-Sambucus nigra lectin (SNA) or afibronectin-antibody complex, and/or by detecting and/or quantifyingglycosylated pregnancy-specific glycoprotein (PSG), for example bydetection/quantification of pregnancy specific glycoprotein-Aleuriaaurantia lectin (PSG-AAL) or a PSG-antibody complex. In someembodiments, the proteomic profile may also include information on theexpression of adiponectin, sex hormone binding globulin (SHBG),C-reactive protein (CRP), the ratio of human chorionic gonadotropin(hCG) to placental lactogen, or a combination thereof, such as two,three, or all four of adiponectin, SHBG, CRP, and the ratio of hCG toplacental lactogen.

As used herein, the term “proteome” refers to a significant portion ofthe proteins in a biological sample at a given time. Generally speaking,the proteome is fundamentally different from a genome, in that while thegenome is virtually static, the proteome continually changes in responseto internal and external events. Thus, as used herein, the term“proteomic profile” refers to a representation of the expression patternof a plurality of proteins in a biological sample, such as whole blood,plasma, serum, or saliva, at a given time. In various embodiments, aproteomic profile may, for example, be represented as a mass spectrum,but other representations based on any physicochemical or biochemicalproperties of the proteins are also included. Thus, a proteomic profilemay, for example, be based on differences in the electrophoreticproperties of proteins, as determined by two-dimensional polyacrylamidegel electrophoresis (2D-PAGE) or 2-dimensional differential in-gelelectrophoresis (2D-DIGE), and may be represented, for example, as aplurality of spots in a two-dimensional electrophoresis gel. In variousembodiments, differential expression profiles may have importantdiagnostic value, and protein spots may be detected and/or quantified,for example, by mass spectrometry, immunoblotting, ELISA assays, orprotein microarrays. In various embodiments, proteins may be detectedand/or quantified using lateral flow devices, such as for point-of-careuse, as well as spot check colorimetric tests.

In various embodiments, a proteomic profile in accordance with thepresent disclosure may represent or contain information about at least2, 3, 4, 5, 6, 7, 8, 9, 10, or even more proteins. As used herein, theterm “expression signature” refers to a unique protein expressionfeature or motif within a proteomic profile of a biological sample (suchas a test sample) that may differ from a proteomic profile of acorresponding reference sample (e.g., obtained from the same type ofbiological fluid) in a statistically significant manner. For example, invarious embodiments, an “expression signature characteristic ofgestational diabetes” may include a characteristic motif that differsfrom a proteomic profile of a normal reference sample, or it may share acharacteristic motif with a proteomic profile from a gestationaldiabetes reference sample. In various embodiments, the expressionsignature may include an increase or decrease in the expression of twoor more specific proteins, for example glycosylated fibronectin (forexample, which may be detected as fibronectin-SNA or afibronectin-antibody complex), glycosylated PSG (for example, which maybe detected as PSG-AAL or a PSG-antibody complex), adiponectin, SHBG,and/or CRP, and/or it may include an increase or decrease in the ratioof expression of two or more specific proteins, such as the ratio ofhuman chorionic gonadotropin (hCG) to placental lactogen. For each ofthese proteins, an exemplary GENBANK® Accession number is listed: humanfibronectin, Genbank Accession No. P02751; human pregnancy-specificbeta-1-glycoprotein 9, Genbank Accession No. gi:6093845; human sexhormone binding globulin, Genbank Accession No. gi:134907; humanadiponectin, Genbank Accession No. gi:167077467; humanchoriogonadotropin subunit beta, Genbank Accession No. gi:116184; humanglycoproteins hormone alpha chain, Genbank Accession No. gi:121312;human placental lactogen, Genbank Accession No. gi:190034; and humanC-reactive protein, Genbank Accession No. gi:30224.

In some embodiments, the method includes detecting an increase, such asa statistically significant increase relative to a reference sample,such as at least a 10%, 15%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, oreven greater increase, in the expression of glycosylated fibronectin(which may be detected as, for example, fibronectin-SNA or afibronectin-antibody complex) and glycosylated PSG (which may bedetected as, for example, PSG-AAL or a PSG-antibody complex), or astatistically significant increase in the expression of glycosylatedfibronectin (which may be detected as, for example, fibronectin-SNA or afibronectin-antibody complex) and glycosylated PSG (which may bedetected as, for example, PSG-AAL or a PSG-antibody complex), inaddition to CRP and/or the ratio of hCG to placental lactogen. In someembodiments, the statistically significant increase in the expression ofglycosylated fibronectin and PSG (e.g., fibronectin-SNA or afibronectin-antibody complex and PSG-AAL or a PSG-antibody complex) alsomay be accompanied by a statistically significant decrease, such as atleast a 10%, 15%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, or even greaterdecrease, relative to the reference sample, in expression of adiponectinand/or SHBG. In particular embodiments, an expression signaturecharacteristic of gestational diabetes may include a statisticallysignificant increase in the expression of glycosylated fibronectin(e.g., detected as fibronectin-SNA or a fibronectin-antibody complex),glycosylated PSG (e.g., detected as PSG-AAL or a PSG-antibody complex),CRP, and the ratio of hCG to placental lactogen, in addition to astatistically significant decrease in expression of adiponectin andSHBG.

In various embodiments, if the reference sample is a normal or controlreference sample, such as a sample from a subject who does not havegestational diabetes, and the proteomic profile of the test samplediffers from the reference sample with regard to at least one expressionsignature characteristic of gestational diabetes, then the subject maybe determined to have gestational diabetes. Conversely, if the testsample does not differ from the normal reference sample with regard toat least one expression signature characteristic of gestationaldiabetes, then the subject may be determined to not have gestationaldiabetes. In other embodiments, if the reference sample is a gestationaldiabetes reference sample, such as a sample from a subject who hasgestational diabetes, and the proteomic profile of the test samplediffers from the reference sample with regard to at least one expressionsignature characteristic of gestational diabetes, then the subject maybe determined not to have gestational diabetes. Conversely, if the testsample does not differ from the gestational diabetes reference samplewith regard to at least one expression signature characteristic ofgestational diabetes, then the subject may be determined to havegestational diabetes. Some embodiments may use more than one referencesample, such as a normal reference sample and a gestational diabetesreference sample. Hence, the proteomic profile provides a diagnosticcriterion for gestational diabetes. Statistical methods for determiningif the abundance of a protein of interest is increased or decreasedrelative to a reference sample are well known in the art, and aredescribed below.

In various embodiments, proteomic analysis of biological fluids, such aswhole blood, saliva, or serum, may be performed using a variety ofmethods known to those of skill in the art. In various embodiments, in adirect comparative analysis, the reference sample and test sample may betreated exactly the same way, in order to correctly represent therelative abundance of proteins and obtain accurate results. For example,as discussed above, in various embodiments, the proteins present in thebiological samples may be separated by 2D-gel electrophoresis accordingto their charge and molecular weight. For instance, the proteins mayfirst be separated by their charge using isoelectric focusing(one-dimensional gel electrophoresis), for example using immobilizedpH-gradient (IPG) strips, which are commercially available. In variousembodiments, the second dimension may be an SDS-PAGE analysis, where thefocused IPG strip may be used as the sample. After two-dimensional gelelectrophoresis separation, proteins may then be visualized withconventional dyes, such as Coomassie Blue or silver staining, and imagedusing known techniques and equipment, such as, for example Bio-Rad GS800densitometer and PDQUEST™ software.

In some embodiments, individual spots may then be cut from the gel,de-stained, and subjected to tryptic digestion, allowing the peptidemixtures to be analyzed by mass spectrometry (MS). Alternatively, insome embodiments, the peptides may be separated, for example bycapillary high pressure liquid chromatography (HPLC) and may be analyzedby MS either individually, or in pools. If desired, in some embodiments,the amino acid sequences of the peptide fragments and the proteins fromwhich they derived may be determined. Although it is possible toidentify and sequence all or some of the proteins present in a proteomicprofile, this typically is not necessary for the diagnostic use of theproteomic profiles.

As discussed above, in various embodiments, a diagnosis of gestationaldiabetes may be based on characteristic similarities or differences(e.g., expression signatures) between a reference sample and a testsample. For example, in various embodiments, if the proteomic profile ispresented in the form of a mass spectrum, the expression signature maybe a peak or a combination of peaks that differ, qualitatively orquantitatively, from the mass spectrum of a corresponding normal sample.Thus, the appearance of a new peak or a combination of new peaks in themass spectrum, or any statistically significant change in the amplitudeor shape of an existing peak or combination of existing peaks, or thedisappearance of an existing peak in the mass spectrum may be consideredan expression signature.

Statistical methods for comparing proteomic profiles are well known inthe art. For example, in various embodiments employing a mass spectrum,a proteomic profile may be defined by the peak amplitude values at keymass/charge (M/Z) positions along the horizontal axis of the spectrum.Accordingly, in various embodiments, a characteristic proteomic profilemay, for example, be characterized by the pattern formed by thecombination of spectral amplitudes at given M/Z vales. In variousembodiments, the presence or absence of a characteristic expressionsignature, or the substantial identity of two profiles, may bedetermined by matching the proteomic profile of a test sample with theproteomic profile of a reference sample with an appropriate algorithm.

Other embodiments may utilize protein arrays to monitor proteinexpression levels, enabling high-throughput analysis. Protein arrays areknown to those of skill in the art, and generally are formed byimmobilizing proteins, such as antibodies specific for proteins ofinterest, on a solid surface, such as glass, silicon, nitrocellulose, orPVDF using any of a variety of covalent and non-covalent attachmentchemistries well known in the art. The arrays may be probed withfluorescently labeled proteins from two different sources, such asnormal and test samples, and fluorescence intensity may reflect theexpression level of a target protein.

Various embodiments also may use any of various immunoassay formats forquantification of protein expression levels. In general, immunoassaysmay be homogeneous or heterogeneous. For instance, in variousembodiments, an enzyme-linked immunosorbant assay (ELISA) may be used toquantify protein expression. In one example, in a “sandwich” assay, asolid surface may be coated with a solid phase antibody, and the testsample may be allowed to react with the bound antibody. Any unboundantigen (e.g., protein of interest) may then be washed away, and a knownamount of enzyme-labeled antibody may then be reacted. The label maythen be quantified as a direct measurement of the amount of protein ofinterest present in the sample.

In some embodiments, ELISA may also be used as a competitive assay. Forexample, in a competitive assay, the test sample containing the proteinof interest may be mixed with a precise amount of enzyme-labeled proteinof interest, and both may compete for binding to an antibody attached toa solid surface. In various embodiments, excess free enzyme-labeledprotein may be washed off before the substrate for the enzyme is added,and the color intensity resulting from the enzyme-substrate interactionmay be used as a measure of the amount of protein of interest in thetest sample.

Various other embodiments may quantify the proteins of interest using anEnzyme Multiplied Immunoassay Technique (EMIT), which may include a testsample, enzyme-labeled molecules of the proteins of interest, antibodiesspecific to the proteins of interest, and a specific enzyme chromogenicsubstrate. In various embodiments, an excess of the specific antibodiesmay be added to the test sample, and the proteins of interest may thenbind to the antibodies. In various embodiments, a measured amount of thecorresponding enzyme-labeled proteins may then be added to the mixture,and antibody binding sites not occupied by proteins of interest from thetest sample may be occupied with molecules of the enzyme-labeledprotein. As a result, in various embodiments, enzyme activity may bereduced because only free enzyme-labeled protein can act on thesubstrate, and the amount of converted substrate may reflect the amountof free enzyme left in the mixture. In various embodiments, a highconcentration of the protein of interest in the sample may result inhigher absorbance readings.

Various other embodiments include immunoassay kits for thequantification of the proteins of interest in a test sample. In variousembodiments, these kits may include, in separate containers, monoclonalantibodies having binding specificity for each of the proteins ofinterest, and, optionally, anti-antibody immunoglobulins, particularlylabeled anti-antibody immunoglobulins.

Also disclosed herein are capture devices and sample collection kits foruse in the disclosed methods. In some embodiments, the disclosed methodsmay be carried out using a sample capture device, such as a lateral flowdevice (for example a lateral flow test strip) that may allowquantification of two or more proteins of interest. Lateral flow devicesare available in numerous different configurations, but in one example,a test strip may include a flow path from an upstream sample applicationarea to a test site, such as from a sample application area through amobilization zone to a capture zone. In various embodiments, themobilization zone may contain a mobilizable marker that may interactwith the protein of interest, and the capture zone may contain a reagentthat binds the protein of interest for detection and/or quantification.In other embodiments, exemplary sample collection kits may include anabsorbent medium, such as filter paper, that may include indicia for theplacement of the test sample on the medium. Such kits also may include alancing device for obtaining a blood sample from a subject, andoptionally, a mailer for sending the test sample to a physician orlaboratory for analysis. Such sample collection kits may be used, forexample, during standard prenatal exams, such as the twelve, sixteen,twenty, or twenty-four-week visit, and/or sample collection may beperformed when blood is obtained for other standard prenatal tests.

The following Examples are provided for illustration purposes, and arenot to be construed as limiting in any way.

EXAMPLES Example 1 Subject Selection

A total of 1463 consecutive women within the second and third trimestersof pregnancy underwent a 75-gram OGTT followed by a 2-hour plasmaglucose determination. GDM was diagnosed as a 2-hour plasma glucose>7.8mmol/L, consistent with WHO criteria. All remaining women werecategorized as non-diabetic.

Fourteen non-diabetic and 15 GDM participants were randomly selectedfrom the described population. Clinical characteristics of participantsare described in Table 1.

TABLE 1 Participant characteristics by GDM status Study Group (n)Participant Characteristic Non-diabetic Gestational Mean (SD) (14)Diabetes (15) p-value¹ Age (years) 24.2 (3.7) 24.6 (3.5) 0.78Pre-pregnancy Body 19.6 (3.4) 20.9 (3.2) 0.32 Mass Index (kg/m²) Percentweight change 15.2 (4.9) 14.2 (3.7) 0.54 (%) Blood Pressure (mmHg)Systolic 106 (11) 110 (11) 0.38 Diastolic 71 (6) 69 (7) 0.45 TotalCholesterol (mg/dl) 213 (44) 190 (28) 0.11 Low-density lipoprotein 120(41) 104 (30) 0.23 (mg/dl) Very-low-density 42 (10) 38 (13) 0.4lipoprotein (mg/dl) High-density lipoprotein 51 (3) 49 (4) 0.11 (mg/dl)Median (IQR) Triglycerides (mg/dl) 222 (178, 238) 189 (133, 227) 0.36Fasting plasma glucose 80 (77, 85) 84 (79, 90) 0.2 (mg/dl) GlycatedHemoglobin 5.2 (5.0, 5.4) 5.4 (5.1, 5.8) 0.23 (%) C-peptide (ng/ml) 0.9(0.6, 1.2) 0.7 (0.4, 1.1) 0.25

Example 2 Proteomic Profiles

Serum samples were analyzed to obtain measures of sex-hormone bindingglobulin (SHBG), adiponectin, human chorionic gonadotropin (hCG),placental lactogen, C-reactive protein (CRP), pregnancy specificglycoprotein-1 (PSG-1), and fibronectin, as well as specificglycosylated forms of fibronectin and PSG-1 (Table 2). Two-dimensionaldifferential in-gel electrophoresis (2D-DIGE) and immunoassays (ELISA)were performed.

TABLE 2 Differences in serum analyte levels between normal andgestational diabetic pregnant women Protein Study Group ConcentrationNon-diabetic Gestational Diabetes Median (IQR) (14) (15) p-value¹C-Reactive Protein 2.1 (1.0, 4.0) 5.7 (2.2, 9.0) 0.05 (mg/L) SHBG (mg/L)276 (252, 304) 240 (173, 278) 0.12 Adiponectin 4.1 (3.3, 5.0) 3.4 (2.3,5.2) 0.28 (mg/mL) hCG/Placental 1.6 (1.0, 2.3) 3.2 (1.6, 4.3) 0.03Lactogen Ratio PSG-1 (AU) 1.16 (0.96, 1.52) 1.21 (0.80, 1.40) 0.95Fibronectin (mg/L) 96.0 (78.8, 151.9) 151.5 (55.4, 238.6) 0.33 ProteinGlycosylation PSG-AAL (AU/mL) 52.5 (46.7, 71.0) 85.7 (69.9, 99.5) 0.004Fibronectin-SNA 51.0 (45.8, 55.1) 67.0 (53.5, 84.0) 0.006 (AU/mL)

Differential glycosylation of fibronectin and PSG-1 was determined bydirect lectin binding immunoassays. T-tests were used for analysis ofnormally distributed continuous variables and the Wilcoxon nonparametricequivalent for variables with skewed distribution. Chisquare andFisher's Exact tests were used for categorical variables. Parametric andWilcoxon nonparametric t-tests were used to test differences acrossgroups for variables with normal and skewed distributions, respectively.Ratios of proteins were computed and tested across study groups usingWilcoxon nonparametric t-tests.

Receiver Operating Characteristic (ROC) curves generated from predictedprobabilities from logistic regression modeling were used to evaluatethe classification ability of individual and multiple analytecombinations. The area under the ROC curve (AUROC) was computed fromsimple logistic regression to describe the classification ability ofeach protein, ratio, and glycosylated protein individually. Based on theAUROC results, individual proteins, ratios, and glycosylated proteinwere added sequentially to build a multi-analyte model for improvedclassification performance. All statistical analyses were performedusing SAS software version 9.22 (SAS Institute Inc., Cary, N.C.).

Example 3 Identification of Expression Signature Characteristic ofGestational Diabetes

FIG. 1 illustrates a 2D-DIGE comparison of the total glycoproteinfraction of pooled control and GDM maternal serum, in which proteinspots that were differentially abundant in control vs. GDM samplesappear as green or red spots, while proteins present at similar levelsappear as yellow, in accordance with various embodiments. The arrowspoint to individual protein spots that correspond to differentiallyabundant putative biomarkers.

Two specific maternal serum glycoproteins were selected, fibronectin andpregnancy specific glycoprotein (PSG-1), for assessment of potentialchanges in glycosylation status. Lectin reactivity profiling revealedthat fibronectin glycosylation associated with Sambucus nigra lectin(SNA) binding and PSG-1 glycosylation associated with Aleuria aurantialectin (AAL) binding were significantly elevated in GDM maternal serumvs. control serum. Therefore, these two protein-lectin pairs,fibronectin-SNA and PSG-AAL, were selected for inclusion in amulti-analyte panel with additional biomarkers previously demonstratedto exhibit differential abundance in GDM, including adiponectin, sexhormone binding globulin (SHBG), and C-reactive protein (CRP), as wellas the ratio of human chorionic gonadotropin (hCG) to placentallactogen. These analytes were evaluated singly and in combination in aset of control and GDM maternal serum samples from the cohort describedin Table 1.

The mean participant age and pre-pregnancy BMI were 24.4+3.5 years and20.3+3.3 kg/m2, respectively. Glycated hemoglobin measures did notdiffer between non-diabetic and GDM participants [5.2%(IQR: 5.0-5.4%)and 5.4%(IRQ: 5.1-5.8%), respectively]. Fasting plasma glucose measureswere also similar between groups [80 mg/dl (IQR: 77-85 mg/dl) and 84mg/dl (IQR: 79-90 mg/dl); p=0.20]. In addition, there was nostatistically significant difference observed between study groups withregard to any other clinical parameter that was measured.

As shown in Table 2, the levels of PSG-AAL, fibronectin-SNA and thehCG/placental lactogen ratio were significantly elevated in the GDMgroup (p=0.004, p=0.006 and p=0.03, respectively). The difference inmaternal serum CRP levels demonstrated borderline significance (p=0.05),with a median concentration of 2.1 mg/L in non-diabetics and 5.7 mg/L inGDM participants. Thus, combining these proteins in a ratio improvesdiscrimination ability.

FIG. 2 illustrates receiver operating characteristic (ROC) curves thatdemonstrate the ability of fibronectin and pregnancy-specificglycoprotein (PSG) glycosylation to distinguish samples from pregnantwomen with and without gestational diabetes, in accordance with variousembodiments. FIG. 3 illustrates ROC curves that demonstrate theclassification performance of each protein and protein glycosylationpattern as individual analytes and as a multi-analyte model; inaccordance with various embodiments. While the ability to detect GDMusing expression of the two glycosylated proteins PSG-AAL andfibronectin-SNA alone is good (AUROC: 0.85; FIG. 2), their use inconjunction with the other analytes described in Table 2 within amulti-analyte model (FIG. 3) demonstrated clearly superior performance(AUROC: 0.97). Specifically, the combination of fibronectin-SNA andPSG-AAL alone had a detection rate of 74% at a false positive rate of 6%(FIG. 2), while the multi-analyte model had a marked increase in thedetection rate (87%) at a false positive rate<1% (FIG. 3).

Thus, a multi-analyte test profile comprised of individual proteins,their ratios, and specific protein glycosylation patterns in maternalserum can identify GDM patients independently classified by OGTT. Theseanalytes are all amenable to analysis in dried blood spots, whichenables the use of a minimally invasive, convenient, and cost-efficientscreening test for GDM that may be particularly useful for evaluation ofunderserved populations that suffer significant disparities in diabetescare.

Example 4 Materials and Methods for First-Trimester Serum Panel forPrediction/Detection of GDM

Plasma glucose was determined by the hexokinase method using a Konelab60i Clinical Chemistry Analyzer (Thermo Electron Co, Finland).

Sources (catalog number) and working dilutions of primary antibodiesused in ELISAs were as follows: SHBG MAb (31401) from Abcam at 1:250;placental lactogen MAb (L1022-03G) from US Biologicals at 1:250; hCG MAb(MAB605) from Millipore at 1:250; fibronectin MAb (MAB1918) at 1:1000,CRP polyclonal Ab (842676) at 1:180, adiponectin MAb (840965) at 1:180,and PAPPA-2 MAb (MAB1668) at 1:500 from R&D systems; and AFP polyclonalAb (A0008) at 1:1000 and PAPPA-1 polyclonal Ab (A0230) at 1:1000 fromDako.

Levels of protein analytes were determined by sandwich ELISA. Primarycoating antibodies were resuspended in carbonate buffer (pH 9.6) and 100μl added to each well of a 96-well Reactibind plate (Pierce) andincubated at 4° C. overnight. Plates were blocked with 3% BSA in PBS (pH7.2). Following sample addition and incubation for 45 minutes at roomtemperature, plates were washed with PBST using a Biotek plate washer,and then incubated with detection antibodies for 45 minutes at roomtemperature. Plates were again washed with PBST and then incubated withstreptavidin-HRP (50 ng/ml in PBS; Pierce) for 45 minutes at roomtemperature, and then washed with PBST. TMB substrate (Neogen) was addedand, following the development of the signal, quenched by the additionof 2N H₂SO₄. The plate was read using an EPOCH plate reader (Biotek) at490 nm, and data was processed using Gen5 software version 1.10.8. Datawas then analyzed as described below in Example 6. A reference samplewas run on each plate. For the serial measures assay run, samples fromall three trimesters for each participant were included on the sameplate. The inter-assay coefficient of variation (CV) for all assays was<10%.

For assay of Fibronectin-SNA, the Fibronectin MAb was used to coatReactibind plates which were then blocked as described above. Theblocking solution was then removed and 200 μl/well of oxidation buffer(100 mM sodium periodate, 50 mM citric acid, pH 4.0) was added and theplate incubated for 14 minutes. The oxidation solution was then removedand the plate washed with PBST before sample addition. Samples wereapplied at a 1:800 dilution, after which the plate was washed with PBST.Biotin-conjugated SNA (Vector Labs) was then added to a finalconcentration of 0.5 ng/μl in PBS. After washing the plate with PBST,the plate was incubated with Streptavidin-HRP and processed as above.The inter-assay CV for FN-SNA was 17%.

Example 5 Subject Population for First-Trimester Serum Panel forPrediction/Detection of GDM

The Finnish Maternity Cohort is a prospective study derived from theserum bank at the National Institute for Health and Welfare (Finland).Participants were recruited from maternity clinics in the area of OuluUniversity Hospital (Oulu, Finland) and Kuopio University Hospital(Kuopio, Finland) between 2004 and 2010. Serum samples were drawn duringregular pre-natal exams, and clinical data was obtained from the BirthRegister, a computerized database containing information on maternalcharacteristics and pregnancy outcome.

The present analysis employed a case-control design, in which a total of90 cases and 92 controls were randomly selected from the describedpopulation. Participants needed a sufficient quantity of 1 st-trimesterserum collected between 5 and 13 weeks gestation for inclusion. EligibleGDM cases included any woman who developed GDM during pregnancy,identified by a standard 75-g OGTT followed by 2-hour plasma glucosedetermination. GDM was diagnosed as a 2-hour plasma glucose>140 mg/dl(7.8 mmol/L), consistent with World Health Organization criteria.Non-diabetic controls were selected from the same population as cases,but did not develop GDM during pregnancy. Maternal characteristics andpregnancy outcome information were extracted from the same database forboth groups. Clinical data was unable to be extracted from the birthregister for 13 non-diabetic Controls. There was no material differencein serum analyte concentration between non-diabetic controls with andwithout clinical data.

In addition to 1st-trimester serum, 35 non-diabetic controls had serumsamples drawn during the 2nd and 3rd trimester. These participants wereincluded in a serial measures analysis to evaluate the change in FN-SNAconcentration across trimester (FIG. 4).

Example 6 Statistical Analysis for First-Trimester Serum Panel forPrediction/Detection of GDM

A thorough descriptive analysis was performed for each maternalcharacteristic and serum analyte measure. Distributions of maternalcharacteristics according to study group were compared using 2-sidedindependent t-tests for continuous variables and Chi-square and Fisher'sexact tests for categorical variables. The Wilcoxon nonparametric t-testwas used for comparisons across groups for continuous variables withskewed distributions. In participants with serial measures offibronectin-SNA, least square means by trimester were computed andcompared using mixed models. The serial measures of fibronectin-SNA wereplotted and repeatability was quantified using a two-way, mixed effectintraclass correlation coefficient.

The independent association between each serum analyte and subsequentdevelopment of GDM was evaluated using univariate and multivariatelog-binomial regression. Risk ratios (RR) and 95% confidence intervals(CI) may be derived from this method using a poisson distribution withrobust variance estimation. Serum analytes were categorized intotertiles and evaluated in univariate and multivariate models. The firstmodel was adjusted for baseline maternal factors including maternal age,nulliparity, and gestational age at sample collection. The second modelincluded all baseline maternal factors in addition to each serum analytesignificantly associated with GDM status (p<0.05). Fibronectin-SNA,adiponectin, hs-CRP, and placental lactogen were included inmultivariate log-binomial regression analyses (FIG. 4).

To evaluate the clinical utility of these serum analytes as individualbiomarkers and as a potential first-trimester panel for prediction ofGDM, Receiver Operating Characteristic (ROC) Curves were generated frompredicted probabilities from logistic regression modeling. The areaunder the ROC curve (AUROC) and 95% confidence interval (CI) wascomputed from simple logistic regression to describe the GDMclassification ability of each serum analyte individually. Amulti-analyte GDM screening panel comprised of fibronectin-SNA,adiponectin, hs-CRP, and placental lactogen was assessed to determinethe discrimination of the multi-analyte model compared to singleanalytes. A backward-selection process was utilized to evaluate thecontribution of each analyte to the multi-analyte panel and determine ifserum analytes were additive. Additional ROC curves were generated todetermine if the classification performance of the multi-analyte panelvaried by maternal parity or time to GDM diagnosis.

Two-sided p-values are reported, and a value of less than 0.05 wasconsidered statistically significant. All statistical analyses wereperformed using SAS software version 9.3 (SAS Institute Inc., Cary,N.C.).

Example 7 2D-DIGE for First-Trimester Serum Panel forPrediction/Detection of GDM

GDM was associated with changes in the GDM maternal serum glycoproteomeas assessed by 2D-DIGE. This is illustrated in FIG. 1, which shows a2D-DIGE comparison of the total glycoprotein fraction of pooled controland GDM maternal serum, in which protein spots that were differentiallyabundant in control vs. GDM samples appear as green or red spots, whileproteins present at similar levels appear as yellow. The arrows point toindividual protein spots that correspond to differentially abundantcandidate biomarkers.

In subsequent analyses, fibronectin was selected for assessment ofpotential changes in glycosylation status. Lectin reactivity profilingrevealed that fibronectin glycosylation associated with Sambucus nigralectin (SNA) binding (FN-SNA) was significantly elevated in1st-trimester GDM maternal serum vs. control serum. Therefore, FN-SNAwas included in a multi-analyte panel with additional biomarkerspreviously demonstrated to exhibit differential abundance in GDM,including adiponectin, sex hormone binding globulin (SHBG), andC-reactive protein (CRP), as well as the ratio of human chorionicgonadotropin (hCG) to placental lactogen. These analytes were evaluatedsingly and in combination in a set of control and GDM maternal serumsamples from the cohort described in Table 3, below.

TABLE 3 First-trimester serum protein and protein glycosylationconcentration by gestational diabetes status. Study Group (n) MaternalNon-diabetic Gestational Characteristic Controls Diabetes Mean (SD) (92)(90) p-value¹ Maternal age (years) 26.2 (4.0) 31.3 (6.0) <0.0001Gestational age at sample 9.7 (1.4) 10.1 (0.8) 0.009 collection (weeks)Gestational age at delivery 40.2 (1.1) 39.7 (1.6) 0.03 (weeks)² Infantbirthweight (g)² 3563 (401) 3644 (512) 0.26 Number (%) Nulliparity² 62(82) 29 (32) <0.0001 Macrosomia (>4000 g)² 12 (16) 20 (22) 0.36 SerumAnalyte Concentration Mean (SD) Fibronectin-SNA (mg/L) 80 (35) 132 (36)<0.0001 Adiponectin (μg/ml) 3.0 (1.2) 2.5 (0.9) 0.001 SHBG (nmol/L) 91(66) 84 (46) 0.43 Median (IQR) C-reactive protein (mg/L) 0.39 (0.17,1.24) 1.17 (0.52, 2.04) <0.0001 Placental Lactogen (mg/L) 0.22 (0.05,0.36) 0.34 (0.23, 0.63) <0.0001 hCG (mIU/ml) 60911 (50655, 72984) 61542(48287, 70467) 0.65 ¹Comparisons were made using a 2-sided independentt-test and Wilcoxon 2-sided nonparametric t-test for continuousvariables. Chi-square and Fisher's exact tests were used for categoricalvariables. ²Gestational age at delivery, birthweight, and parity datawas unavailable for 20, 19, and 16 non-diabetic controls, respectively.

Example 8 First-Trimester Case-Control Analysis

The mean gestational age at sample collection was 9.9+1.2 weeks (range:5.7-13.1) and was not markedly different between GDM and non-diabeticcontrol participants (10.2+0.8 weeks and 9.7+1.4 weeks, respectively).GDM participants were older (31.4+5.9 years vs. 26.2+4.0 years;p<0.0001) and less likely to be nulliparous (34% vs. 82%; p<0.0001) thannon-diabetic controls (Table 3). In GDM participants, the meangestational age at diagnosis was 22.2+6.2 weeks with an average of12.6+6.0 weeks between the 1st-trimester serum draw and GDM diagnosis.

First-trimester concentrations of FN-SNA, adiponectin, hs-CRP, andplacental lactogen were significantly associated with GDM status(p<0.001; Table 3). In particular, FN-SNA demonstrated markedly higherconcentrations in GDM compared to non-diabetic controls, with minimaloverlap in group distributions [mean (95% CI): 132 (124, 139) mg/L vs.80 (72, 87) mg/L, respectively; p<0.0001].

The risk of subsequent development of GDM increased with increasingtertile for FN-SNA, hs-CRP, and placental lactogen (Table 4, below).Adiponectin demonstrated significantly greater risk of GDM if1st-trimester concentrations were below the first tertile (<2.2 μg/ml),but no significant difference in risk ratios between the 2nd and 3rdtertiles (p=0.15). Multivariate analyses demonstrated marked attenuationof risk ratios for all serum analytes upon adjustment for maternal age,nulliparity, and gestational age at sample collection. Despite this,FN-SNA had a strong independent association with GDM after adjustmentfor these maternal factors. In addition, when FN-SNA concentrations<80mg/L were used as the referent group, risk ratios increased between the2nd and 3rd tertile [RR (95% CI): 4.81 (1.85, 12.49) and 7.62 (2.97,19.58), respectively]. The difference between the 2nd and 3rd tertileswas statistically significant (p<0.0001). Upon addition of all serumanalytes to the maternal factors model, there was minimal change inFN-SNA risk ratios. In this fully adjusted model, only FN-SNA andplacental lactogen demonstrated significant independent associationswith development of GDM in this population.

TABLE 4 Risk of subsequent gestational diabetes across tertiles offirst- trimester serum protein and protein glycosylation concentration.Multivariate Analysis Maternal factors + Univariate Analysis Maternalfactors¹ serum analytes RR (95% CI)² p-value RR (95% CI)² p-value RR(95% CI)² p-value Fibronectin- SNA (mg/L) >122    13.77 (5.33, 35.60)<0.0001 7.62 (2.97, 19.58) <0.0001 7.22 (2.86, 18.21) <0.0001  80-122 7.38 (2.77, 19.67) <0.0001 4.81 (1.85, 12.49) 0.001 4.69 (1.85, 11.90)0.001 <80     1.00 1.00 1.00 C-reactive protein (mg/L) >1.30  2.24(1.47, 3.42) 0.0002 1.41 (0.96, 2.09) 0.08 1.03 (0.76, 1.40) 0.850.34-1.30  1.69 (1.07, 2.68) 0.02 1.11 (0.73, 1.67) 0.62 0.99 (0.72,1.35) 0.94 <0.34  1.00 1.00 1.00 Placental lactogen (ng/ml) >0.37  2.31(1.49, 3.60) 0.0002 1.51 (1.00, 2.30) 0.05 1.43 (1.05, 1.94) 0.020.20-0.37  1.91 (1.20, 3.04) 0.006 1.63 (1.10, 2.39) 0.01 1.46 (1.08,1.98) 0.01 <0.20  1.00 1.00 1.00 Adiponectin (μg/ml) >3.1   1.00 1.001.00 2.2-3.1  1.36 (0.90, 2.08) 0.15 1.18 (0.84, 1.68) 0.34 1.13 (0.85,1.51) 0.41 <2.2   1.76 (1.19, 2.58) 0.004 1.33 (0.96, 1.83) 0.09 1.30(0.99, 1.70) 0.06 ¹Adjustment for maternal factors includes maternalage, gestation age at sample

Since statistically significant associations with a disease or outcomedo not always translate into strong diagnostic accuracy, the clinicalutility of these tests was quantified using ROC analyses. The ROC curveis a plot of the sensitivity of a diagnostic test across all possiblefalse-positive fractions (1-specificity; FIG. 5). The AUROC is astatistic that can be used to compare the classification performance ofdiagnostic tests. An AUROC of 1.00 reflects perfect discriminationbetween the groups under study while an AUROC of 0.50 indicates nodiscrimination. As illustrated in Table 5, below, FN-SNA alone had anAUROC of 0.91 (95% CI: 0.87, 0.96). This translates to detection rate of82% of GDM cases from 1st-trimester serum screening at a false-positiverate of 10%. Despite strong univariate associations with GDM status,hs-CRP, adiponectin, and placental lactogen demonstrated only marginalclassification performance. To test whether combining these tests into amulti-analyte model improved diagnostic accuracy, the AUROC was computedfor FN-SNA, adiponectin, hs-CRP, and placental lactogen. Themulti-analyte model yielded an AUROC of 0.91 (95% CI: 0.88, 0.96), whichwas not significantly different than the performance of FN-SNA alone(p=0.48).

TABLE 5 Classification performance of each protein and proteinglycosylation as a first-trimester individual test and as amulti-analyte panel. Sensitivity at fixed false-positive fractionsScreening Test AUROC (95% CI) 5% 10% 15% 20% Fibronectin-SNA 0.91 (0.87,0.96) 64 82 85 94 C-reactive protein 0.68 (0.60, 0.76) 6 18 28 46Placental lactogen 0.67 (0.59, 0.75) 7 16 27 42 Adiponectin 0.63 (0.55,0.71) 12 30 35 38 Multi-analyte model 0.92 (0.88, 0.96) 66 78 85 92Fibronectin-SNA C-reactive protein Adiponectin Placental Lactogen ¹Areaunder the Receiver Operating Characteristic Curve (AUROC) and 95% CIwere obtained using univariate and multivariate logistic regressionmodeling.

Stratification by key maternal characteristics was performed todetermine if the classification performance was influence by thesefactors. Upon stratification for nulliparity, no difference was observedin the AUROC of FN-SNA. In addition, the time between 1st-trimesterscreening and diagnosis of GDM may influence the diagnostic accuracy ofthe test if FN-SNA can only detect current or imminent GDM. When theanalysis was restricted to cases with a GDM diagnosis occurring morethan 10 weeks since their 1st-trimester sample collection, the FN-SNAAUROC was similar [AUROC (95% CI): 0.92 (0.87, 0.96)]. Furthermore, nocorrelation was observed between FN-SNA concentration and time between 1st-trimester sample collection and GDM diagnosis (r=−0.06).

Example 9 Fibronectin-SNA Serial Measures

The intraclass correlation coefficient demonstrated modest FN-SNArepeatability across trimester in non-diabetic controls with repeatedserum measures (r=0.44). A significant difference in FN-SNAconcentration was observed across the first, second, and thirdtrimesters [mean (95% CI): 66 (59, 72) mg/L, 56 (50, 63) mg/L and 69(62, 75) mg/L, respectively; p=0.003]. There was no significantdifference between the first and third trimester with regard to FN-SNAconcentration (p=0.51). Despite change across trimesters, only 4non-diabetic control participants had FN-SNA concentrations above the25th percentile of the 1st-trimester GDM population.

These analyses illustrate the utility of a panel comprised of individualprotein analytes, analyte ratios, and specific protein glycosylation inmaternal serum to efficiently identify GDM patients independentlyclassified by OGTT. The robust performance of FN-SNA alone in both 1st,2nd, and 3rd trimesters, coupled with its enhanced 1st-trimesterperformance with the addition of CRP, SHBG, and the hCG/placentallactogen ratio, supports the use of this panel for both early screeningand subsequent confirmation/diagnosis of GDM. In fact, the performanceof fibronectin-SNA alone in terms of specificity and sensitivity,surpasses that of the 50 g glucose challenge test which has beenproposed as a screening precursor to the standard OGTT. These analytesare all amenable to analysis in dried blood spots, which enables the useof a minimally invasive, convenient, and cost-efficient screening testfor GDM that is particularly useful for evaluation of underservedpopulations that suffer significant disparities in diabetes care.

The majority of the components of the multianalyte panel, in addition tofibronectin-SNA itself, are also glycoproteins, and their glycosylationhas been reported to be altered in pathological conditions to modulateserum stability or regulate activity. Thus, their utility inidentification of GDM further supports the notion that the glycoproteomeconstitutes an important class of biomarkers that is intimatelyconnected to cellular function and pathophysiology.

Example 10 Serum Markers for Diagnosis of GDM

A total of 1463 consecutive women within the second and third trimesterof pregnancy underwent a 75 g oral glucose tolerance test (OGTT)followed by a 2-hour plasma glucose (PG). Gestational diabetes (GDM) wasdiagnosed as a 2-hour PG>7.8 mmol/L, consistent with WHO criteria. Allremaining women were categorized as non-diabetic.

A case-control design was used in which 14 non-diabetic and 15 GDMparticipants were randomly selected from the described population. Bloodsamples from the OGTT were used to obtain measures of sex-hormonebinding globulin (SHBG), adiponectin, human chorionic gonadotropin(hCG), placental lactogen, C-reactive protein (CRP), PSG-1, andfibronectin as well the glycosylated form of fibronectin and PSG-1.

Differential glycosylation of specific proteins (e.g., fibronectin andPSG-1) was determined by direct lectin binding assay, in which proteinswere immunoprecipitated from maternal serum. Maternal serum levels oftotal fibronectin and PSG-1, adiponectin, CRP, hCG, and placentallactogen were assayed by commercial ELISA kits.

A thorough descriptive analysis was performed for each protein,glycosylated protein and participant characteristic. Parametric andWilcoxon nonparametric t-tests were used to test differences acrossstudy groups for variables with a normal and skewed distribution,respectively. Ratios of proteins were computed and tested across studygroups using Wilcoxon nonparametric t-tests.

Receiver Operating Characteristic (ROC) Curves generated from predictedprobabilities from logistic regression modeling can be used to evaluatethe classification ability of individual and multiple analytecombinations. The area under the ROC curve (AUROC) was computed fromsimple logistic regression to describe the classification ability ofeach protein, ratio, and glycosylated protein individually. Based onAUROC results, individual proteins, ratios, and glycosylated proteinwere added sequentially to build a multi-analyte model for improvedclassification performance.

All statistical analyses were performed using SAS software version 9.22(SAS Institute Inc., Cary, N.C.).

Characteristics of the sample population are described in Table 6,below. The mean participant age and pre-pregnancy BMI were 24.4+3.5years and 20.3+3.3 kg/m2, respectively. Glycated hemoglobin measures didnot differ between non-diabetic and GDM participants [5.2%(IQR:5.0-5.4%) and 5.4%(IRQ: 5.1-5.8%), respectively]. Fasting plasma glucosemeasures were also similar between groups [80 mg/dl (IQR: 77-85 mg/dl)and 84 mg/dl (IQR: 79-90 mg/dl); p=0.20]. In addition, there was nostatistically significant difference observed between study groups withregard to any other clinical parameter that was measured.

TABLE 6 Participant characteristics by gestational diabetes status StudyGroup (n) Gestational Participant Characteristic Non-diabetic Diabetesp- Mean (SD) (14) (15) value¹ Age (years) 24.2 (3.7) 24.6 (3.5) 0.78Pre-pregnancy body mass 19.6 (3.4) 20.9 (3.2) 0.32 index (kg/m²) Percentweight change 15.2 (4.9) 14.2 (3.7) 0.54 (%) Blood pressure (mmHg)Systolic 106 (11) 110 (11) 0.38 Diastolic 71 (6) 69 (7) 0.45 Totalcholesterol (mg/dl) 213 (44) 190 (28) 0.11 Low-density lipoprotein 120(41) 104 (30) 0.23 (mg/dl) High-density lipoprotein 51 (3) 49 (4) 0.11(mg/dl) Median (IQR) Triglycerides (mg/dl) 222 (178, 238) 189 (133, 227)0.36 Fasting plasma glucose 80 (77, 85) 84 (79, 90) 0.20 (mg/dl)Glycated hemoglobin 5.2 (5.0, 5.4) 5.4 (5.1, 5.8) 0.23 (%) C-peptide(ng/ml) 0.9 (0.6, 1.2) 0.7 (0.4, 1.1) 0.25 ¹T-tests for normallydistributed continuous variables and the Wilcoxon nonparametricequivalent for variables with a skewed distribution. Chi-square andFisher's Exact tests were used for categorical variables.

As shown in Table 7, below, the levels of PSG-AAL and fibronectin-SNAwere each significantly elevated in the GDM group, as was thehCG/placental lactogen ratio. While hCG and placental lactogen have beenreported to exhibit altered levels in GDM maternal serum, their ratiowas clearly highly discriminant in this sample set.

TABLE 7 Protein concentration and glycosylation by gestational diabetesstatus Protein Study Group (n) Concentration Non-diabetic GestationalDiabetes Median (IQR) (14) (15) p-value¹ C-Reactive Protein 2.1 (1.0,4.0) 5.7 (2.2, 9.0) 0.05 (mg/L) SHBG (nmol/L) 276 (252, 304) 240 (173,278) 0.12 Adiponectin 4.1 (3.3, 5.0) 3.4 (2.3, 5.2) 0.28 (μg/mL)hCG/Placental 1.6 (1.0, 2.3) 3.2 (1.6, 4.3) 0.03 Lactogen Ratio PSG-1(AU) 1.16 (0.96, 1.52) 1.21 (0.80, 1.40) 0.95 Fibronectin (mg/L) 96.0(78.8, 151.9) 151.5 (55.4, 238.6) 0.33 Protein Glycosylation (arbitraryunits) PSG-AAL 52.5 (46.7, 71.0) 85.7 (69.9, 99.5) 0.004 Fibronectin-SNA51.0 (45.8, 55.1) 67.0 (53.5, 84.0) 0.006 ¹Wilcoxon nonparametric t-test

Receiver-operator characteristics (ROC) curves utilizing fibronectin-SNAand PSG-AAL as well as the combination are shown in FIG. 6. While thesensitivity and specificity of these two analytes for detecting GDM isgood, the additional of the additional analytes in Table 7 in amulti-analyte model (FIG. 7) demonstrated clearly superior performance,even though these analytes themselves were not significantlydifferentially abundant individually in non-diabetic vs. GDM maternalserum.

Example 11 Fibronectin Lateral Flow Immunoassay (FN LFIA)

Various lateral flow assay methods may be utilized to test for thepresence or absence or quantity of an analyte in a biological sample. Inone example, a “sandwich” assay method uses an antibody immobilized on asolid support, which forms part of a complex with a labeled antibody, todetermine the presence of a target analyte by observing the presence andamount of bound analyte-labeled antibody complex. For the purposes of alateral flow immunoassay, the label may be an enzyme, coloredmicrospheres, fluorescently-labeled microspheres, or may use othersimilar detection methods that provide for detection and/orquantification of analyte bound to the test line.

Conventional lateral flow test strips feature a solid support on whichthe sample receiving area and the target capture zones are supported.The solid support material is one which is capable of supporting thesample receiving area and target capture zones and providing for thecapillary flow of sample out from the sample receiving area to thetarget capture zones when the lateral flow test strip is exposed to anappropriate solvent or buffer which acts as a carrier liquid for thesample. General classes of materials that may be used as support includeorganic or inorganic polymers, and natural and synthetic polymers. Morespecific examples of suitable solid supports include, withoutlimitation, glass fiber, cellulose, nylon, crosslinked dextran, variouschromatographic papers and nitrocellulose. One particularly usefulmaterial is nitrocellulose.

FIGS. 8A and 8B illustrate a schematic diagram showing an example of alateral flow immunoassay (FIG. 8A) and a lateral flow test device (FIG.8B) that may be used in accordance with various embodiments disclosedherein. Briefly, 200 μg/mL Rabbit anti-fibronectin was immobilized onthe membrane as a test line (0.5 μL/strip) and 300 μg/mL goat anti-mouseIgG was immobilized as the procedural control line (0.5 μL/strip). Mouseanti-fibronectin-conjugated microspheres (10 μL of 150 μg/mL mouseanti-fibronectin, 1 mg/mL solids) were dried onto a conjugate pad thathad been treated with a solution containing (per liter): 3.81 g SodiumBorate, 2.0 g Dextran, 5.0 g BSA, 1.0 g Tween-20, and 0.5 g SodiumAzide, pH 8.0, followed by drying for 1 hour at 50° C.

The sample was then diluted 1:500 in HEPES Running Buffer (10 mM HEPES,0.1 mM CaCl₂, 155 mM NaCl, 0.1% NaN₃, 0.75% Tween-20, and 0.01%Polyvinyl alcohol). When the sample was applied to the sample pad,capillary flow allowed the fibronectin-containing sample to hydrate andinteract with the labeled microspheres, forming fibronectin-labeledmicrosphere complexes, which further migrated to the test line, wherethey were captured by the rabbit anti-fibronectin.

Following completion of the capillary migration, the device was scanned,and the amount of fibronectin in the sample was determined byquantitative densitometry relative to a standard curve using purifiedfibronectin as standard. FIG. 9 is a graph illustrating a comparison ofthe results of a fibronectin ELISA with the results of the FN-LFIA testdescribed above. As can be seen in FIG. 9, the results of the two testscorresponded with one another quite closely, indicating that the FN-LFIAtest is sensitive and accurate.

Although certain embodiments have been illustrated and described herein,it will be appreciated by those of ordinary skill in the art that a widevariety of alternate and/or equivalent embodiments or implementationscalculated to achieve the same purposes may be substituted for theembodiments shown and described without departing from the scope. Thosewith skill in the art will readily appreciate that embodiments may beimplemented in a very wide variety of ways. This application is intendedto cover any adaptations or variations of the embodiments discussedherein. Therefore, it is manifestly intended that embodiments be limitedonly by the claims and the equivalents thereof.

What is claimed is:
 1. A method for determining if a subject hasgestational diabetes, comprising: providing a test sample from thesubject; measuring a level of glycosylated fibronectin in the sample;determining whether the level of glycosylated fibronectin is elevated inthe sample relative to a control.
 2. The method of claim 1, whereinmeasuring a level of glycosylated fibronectin in the sample comprisesdetecting glycosylated fibronectin using an antibody.
 3. The method ofclaim 2, wherein the antibody is an anti-fibronectin-SNA antibody. 4.The method of claim 7, wherein measuring a level of glycosylated PSG inthe sample comprises detecting glycosylated PSG using an antibody. 5.The method of claim 4, wherein the antibody is an anti-PSG-AAL antibody.6. The method of claim 7, wherein the method further comprises measuringadiponectin, sex hormone binding globulin (SHBG), C-reactive protein(CRP), a ratio of human chorionic gonadotropin (hCG) to placentallactogen, or a combination thereof in the sample.
 7. The method of claim1, the method further comprising: measuring a level of glycosylated PSGin the sample; and determining whether the level of glycosylated PSG iselevated in the sample relative to a control.
 8. The method of claim 6,wherein method further comprises measuring three or more of the groupconsisting of adiponectin, sex hormone binding globulin (SHBG),C-reactive protein (CRP), a ratio of human chorionic gonadotropin (hCG)to placental lactogen, or a combination thereof in the sample.
 9. Themethod of claim 8, wherein method further comprises measuringadiponectin, sex hormone binding globulin (SHBG), C-reactive protein(CRP), and a ratio of human chorionic gonadotropin (hCG) to placentallactogen in the sample.
 10. The method of claim 1, wherein the controlcomprises a reference value.
 11. The method of claim 1, wherein thecontrol is a control reference sample from a non-diabetic subject. 12.The method of claim 7, wherein the method comprises determining whetherthe level of glycosylated fibronectin and the level of glycosylated PSGis elevated in the sample relative to a control.
 13. The method of claim7, wherein the method further comprises determining whether a level ofC-reactive protein (CRP) and/or a ratio of human chorionic gonadotropin(hCG) to placental lactogen is elevated in the sample relative to acontrol.
 14. The method of claim 7, wherein the method further comprisesdetermining whether adiponectin and/or sex hormone binding globulin(SHBG) are decreased in the sample relative to a control.
 15. The methodof claim 7, wherein an elevation relative to the control in the level ofglycosylated fibronectin and/or the level of glycosylated PSG in thesample indicates that the subject has gestational diabetes.
 16. Themethod of claim 15, wherein an elevation relative to the control in thelevel of glycosylated fibronectin and the level of glycosylated PSG inthe sample indicates that the subject has gestational diabetes.
 17. Themethod of claim 16, wherein the method further comprises determiningwhether a level of C-reactive protein (CRP) and/or a ratio of humanchorionic gonadotropin (hCG) to placental lactogen is elevated in thesample relative to a control, and wherein an elevation relative to thecontrol of the level of C-reactive protein (CRP) and/or a ratio of humanchorionic gonadotropin (hCG) to placental lactogen.
 18. The method ofclaim 16, wherein the method further comprises wherein the methodfurther comprises determining whether adiponectin and/or sex hormonebinding globulin (SHBG) are decreased in the sample relative to acontrol, and wherein a decrease relative to control in the level ofadiponectin and/or sex hormone binding globulin (SHBG) indicates thatthe subject has gestational diabetes.
 19. The method of claim 16,wherein the method further comprises determining whether a level ofC-reactive protein (CRP) and a ratio of human chorionic gonadotropin(hCG) to placental lactogen are elevated in the sample relative to acontrol, and determining whether adiponectin and sex hormone bindingglobulin (SHBG) are decreased in the sample relative to a control, andwherein an elevation relative to the control of the level of C-reactiveprotein (CRP) and/or a ratio of human chorionic gonadotropin (hCG) toplacental lactogen, or a decrease relative to control in the level ofadiponectin and/or sex hormone binding globulin (SHBG) indicates thatthe subject has gestational diabetes.
 20. The method of claim 19,wherein the increase or decrease relative to the control is at least10%.
 21. (canceled)
 22. (canceled)
 23. The method of claim 1, whereinthe level of glycosylated fibronectin and/or the level of glycosylatedPSG in the sample is determined using a lateral flow device.
 24. A kitfor carrying out the method of claim 1, the kit comprising an antibodyspecific for glycosylated fibronectin and instructions for using thekit.
 25. A kit for carrying out the method of claim 7, the kitcomprising an antibody specific for glycosylated fibronectin and anantibody specific for glycosylated PSG and instructions for using thekit.